Spontaneous Membranization in a Silk‐Based Coacervate Protocell Model

Abstract Molecularly crowded coacervate micro‐droplets are useful protocell constructs but the absence of a physical membrane limits their application as cytomimetic models. Auxiliary surface‐active agents have been harnessed to stabilize the coacervate droplets by irreversible shell formation but endogenous processes of reversible membranization have received minimal attention. Herein, we describe a dynamic alginate/silk coacervate‐based protocell model in which membrane‐less droplets are reversibly reconfigured and inflated into semipermeable coacervate vesicles by spontaneous self‐organization of amphiphilic silk polymers at the droplet surface under non‐neutral charge conditions in the absence of auxiliary agents. We show that membranization can be reversibly controlled endogenously by programming the pH within the protocells using an antagonistic enzyme system such that structural reconfigurations in the protocell microstructure are coupled to the trafficking of water‐soluble solutes. Our results open new perspectives in the design of hybrid protocell models with dynamical structural properties.

Zeta potential measurements: Zeta potential distributions were used to determine the surface charge of different silk-based polymers (1 mg/mL). Measurements were undertaken at room temperature using a zeta potentiometer analyser (Malvern Instruments, UK). cooled to room temperature. 10 mg of 1,6-hexanediamine and 50 mg EDC were then added to start the amination reaction, which consumed less than 3.5% of the alginate carboxylic acid groups.

Preparation and characterization of silk-based coacervates
The mixture was left at room temperature overnight under stirring and then dialysed against running DI water for 2 days using a Slide-a-Lyzer dialysis cassette (Pierce, MWCO: 12-14 kDa).
Fluorescence labelling of the covalently linked primary amines was undertaken by addition of 1 mL RITC/DMSO or FITC/DMSO (2 mg/mL) to the dialysed cationized alginate solution at room temperature for 6 h under stirring. Fluorescently labelled alginate was purified by dialysis against DI water for 4 days and lyophilization. Typically the FITC/RITC-alginate : alginate mass ratio was 1 :

9.
Methods: Silk-based coacervates were imaged with a Leica DMI3000 B Fluorescence Microscope (Fluo. Microscope, Leica, Germany) and/or a SP5-II Confocal Laser Scanning Microscope (LSCM, Leica, Germany) and analysed using Image J software. Zeta potential distributions were determined on all the different types of silk-based coacervate droplets and vesicles using a zeta potentiometer analyser (Malvern Instruments, UK). The stability of coacervate droplets/vesicles with respect to coalescence in unstirred suspensions or after centrifugation at 1000 rpm for 2 min was monitored by LSCM. Measurements of fluorescence recovery after bleaching (FRAB) were undertaken to assess the fluidity of the silk-based microstructures using a SP5-II Confocal Laser Scanning Microscope (LSCM, Leica, Germany). Secondary structure conformations of CSF in the prepared silk-based coacervate droplets/vesicles were determined by ATR-FTIR spectroscopy using a Nicolet 5700 FTIR (Nicolet Co., USA). The IR absorption spectra were recorded at 600 to 4000 cm -1 .
SAXS experiments were undertaken as follows. Suspensions of positively charged silk-based coacervate vesicles or neutral coacervate micro-droplets were prepared at identical alginate and CSF concentrations (NH2, 4 mM; COOH, 4 mM) but at pH 6 or 8.5, respectively, using additions of NaOH and HCl solution. The suspensions (1 mL) were left unstirred at room temperature for 24 h to facilitate sedimentation/concentration. The concentrated samples were collected after removal of the supernatants and then transferred/sealed in Borokapillaren Mark-Tubes for SAXS. Samples were run in a Q range of 0.005-0.29 Å-1 for 1800 seconds. Data were collected using SAXSGUI instrumentation and analysed using SASView 4.0.

pH-mediated transitions:
Coacervate vesicle-to-coacervate droplet transitions were induced by stepwise addition of aliquots of NaOH or HCl to influence the degree of carboxylate ionisation associated with the changes in pH. Positively charged coacervate vesicles (COOH : NH2 = 1; -COOH, 4mM, -NH2 4 mM, pH 6.5) were prepared as above and the pH increased from 6.5 to 8.0 to 8.3 by gradual addition of NaOH (0.05 M) to produce homogeneous coacervate micro-droplets. The transition was reversed by subsequent six stepwise additions of HCl (0.05 M) to decrease the pH from 8.3 to 7.8 to 7.1 to 6.0 to 4.7 to 3.7 and to 1.3. Each addition of NaOH or HCl was undertaken 30 mins after the previous pH change to provide sufficient time for the pH-induced structural transitions to occur. LSCM images and zeta potential measurements were recorded at each stage of the transitions.
Reversible silk-based coacervate reconfigurations were also achieved by separate additions of urea (500 mM, 2 μL) or glucose (500 mM, 2 μL) to the silk-based vesicles (pH 6.5, 100 μL) or coacervate droplets (pH 8.5, 100 μL) to give a final urea or glucose concentration of ca. 10 mM. Timedependent LSCM images were recorded and quantitatively analysed with the Image J software.  Guest trafficking: Fluorescence dyes (calcein, Rho.123, Rho. 6G, Nile Red) with variable molecular polarities were separately added to a suspension of positively charged coacervate vesicles (alginate 4 mM, -NH2 4 mM, pH: 6.5), followed by stepwise addition of NaOH to increase the pH from 6.5 to 7.5 (positively charged multi-compartmentalized coacervate droplets) to 8.5 (homogeneous coacervate droplets). Resulting changes in the spatial location of the dye molecules within the reconfigured protocells were monitored by LSCM after samples had been held at each pH value for 2 h. Aqueous HCl was then added to decrease the pH from 8.5 to 7.0 (positively charged coacervate vesicles) and LSCM images recorded. Changes in the partition coefficient of the dyes were determined under the different pH conditions. Similar experiments with a range of molecular dyes were undertaken for GOx and urease-loaded silk-based droplets and vesicles undergoing pHinduced reorganization in the presence of glucose (20 mM) or urea (20 mM). Figure S1. Characterization of CSF. (A) Amino group contents of cationized silk fibroin (FITC-CSF, RITC-CSF) and fluorescently labelled silk fibroin (FITC-SF) and unlabeled SF. Primary amines were detected by measuring the UV absorption at 335 nm of CSF and SF solutions after stoichiometric addition of TNBSA and incubation at 37 o C for 2 h. BSA was used to generate a standard curve for the quantitative analysis of -NH2 content in the silk-based polymers. (B) Zeta potential distributions for aqueous SF (red plot) and CSF (green plot) solutions (1 mg/mL). Cationization successfully reverses the surface charge of SF from -9 mV to +25 mV for CSF; data from duplicated samples are shown. (C) NMR spectra of SF (black) and CSF (red); the peak at 1.57 ppm in CSF is the 1 H absorption of βCH2 in 1,6-hexanediamine, indicating that amination between SF and 1,6-hexanediamine occurred. (D) Dynamic light scattering histograms for 1 mg/mL dispersions of SF or CSF. Mean hydrodynamic diameters: 7.5 ± 0.5 nm (SF); 42.5 ± 1.8 nm (CSF).        Experiments with water-insoluble Nile Red were undertaken by dissolving the dye in DMSO (2 mg/mL, 2.5 μL) and mixing with a stock CSF solution (50 μL, [NH2] 8 mM) before or after addition of alginate. The total volume was maintained at 100 μL before or after addition of alginate using DI water (CSF) 4 mM, [COOH] (alginate) 4 mM. Supernatants (50 μL) were mixed with 50 μL Na2CO3/NaHCO3 buffer (100/100 mM, pH 8.5-9.0) for pH manipulation prior to fluorescence emission measurements using a FluoroMax-4 Spectrofluorometer (HORIBA Scientific, Japan). Standard curves were produced by measuring fluorescence emissions of the dyes dissolved in Na2CO3/NaHCO3 buffer (100/100 mM, pH 8.5-9.0) at concentrations of 0.0001, 0.0002, 0.0004, 0.0008 mg/mL). In general, dye concentrations in the corresponding supernatants were lower than their initial concentration (0.0005 mg/mL) in the coacervate suspensions because of coacervate uptake and retention of the molecular cargoes.